Takahisa Maki

The biological membrane is not just a platform for information processing but also a field of mechanics. The lipid bilayer that constitutes the membrane is an elastic body, generating stress upon deformation, while the membrane protein embedded therein deforms the bilayer through structural changes. Among membrane-protein interplays, various channel species act as tension-current converters for signal transduction, serving as elementary processes in mechanobiology. However, in situ studies in chaotically complex cell membranes are challenging, and characterizing the tension dependency of mechanosensitive channels remains semiquantitative owing to technical limitations. Here, we developed a programmable membrane tension-control apparatus on a lipid bilayer system. This synthetic membrane system [contact bubble bilayer (CBB)] uses pressure to drive bilayer tension changes via the Young-Laplace principle, whereas absolute bilayer tension is monitored in real-time through image analysis of the bubble geometry via the Young principle. Consequently, the mechanical nature of the system permits the implementation of closed-loop feedback control of bilayer tension (tension-clamp CBB), maintaining a constant tension for minutes and allowing stepwise tension changes within a hundred milliseconds in the tension range of 0.8 to 15 mN·m^-1. We verified the system performance by examining the single-channel behavior of tension-dependent KcsA and TREK-1 potassium channels under scheduled tension time courses prescribed via visual interfaces. The result revealed steady-state activity and dynamic responses to the step tension changes, which are essential to the biophysical characterization of the channels. The apparatus explores a frontier for quantitative mechanobiology studies and promotes the development of a tension-operating experimental robot.

In eukaryotes, Dmc1 and Rad51 are key proteins of homologous recombination. The Swi5–Sfr1 complex in fission yeast, a conserved auxiliary factor, stimulates DNA strand exchange driven by both Dmc1 and Rad51. Interestingly, biochemical analysis suggested that Swi5–Sfr1 regulates strand exchange activities of these recombinases differently, but the mechanisms were unclear. We previously developed a real-time system to analyze Rad51-driven DNA strand exchange and identified two topologically distinct three-stranded intermediates (complex 1 (C1) and complex 2 (C2)). Swi5–Sfr1 facilitates the C1–C2 transition and releases single-stranded DNA (ssDNA) from C2, acting as a strand exchange activator. In this study, we investigated fission yeast Dmc1-driven DNA strand exchange and the role of Swi5–Sfr1 in Dmc1 activity in real-time. Kinetic analysis revealed a three-step model for the Dmc1-driven reaction, similar to that of Rad51. Although Swi5–Sfr1 stimulated the Dmc1-driven reaction, it had a weaker impact than Rad51. Furthermore, Swi5–Sfr1 enhanced the association of Dmc1 with ssDNA by promoting filament nucleus formation, acting as a mediator, unlike its role with Rad51. This stimulation mechanism also differs from that of Ca2+ or ATP analog, AMP–PNP. Our findings suggest that Swi5–Sfr1 stimulates strand exchange activities of Dmc1 and Rad51 via different reaction steps.

A haploid of the fission yeast Schizosaccharomyces pombe expresses either the P or M mating-type, determined by the active, euchromatic, mat1 cassette. Mating-type is switched by Rad51-driven gene conversion of mat1 using a heterochromatic donor cassette, mat2-P or mat3-M. The Swi2-Swi5 complex, a mating-type switching factor, is central to this process by designating a preferred donor in a cell-type-specific manner. Swi2-Swi5 selectively enables one of two cis-acting recombination enhancers, SRE2 adjacent to mat2-P or SRE3 adjacent to mat3-M. Here, we identified two functionally important motifs in Swi2, a Swi6 (HP1 homolog)-binding site and two DNA-binding AT-hooks. Genetic analysis demonstrated that the AT-hooks were required for Swi2 localization at SRE3 to select the mat3-M donor in P cells, while the Swi6-binding site was required for Swi2 localization at SRE2 to select mat2-P in M cells. In addition, the Swi2-Swi5 complex promoted Rad51-driven strand exchange in vitro. Taken together, our results show how the Swi2-Swi5 complex would localize to recombination enhancers through a cell-type specific binding mechanism and stimulate Rad51-driven gene conversion at the localization site.

A haploid of the fission yeast Schizosaccharomyces pombe expresses either the P or M matingtype, determined by the active, euchromatic, mat1 cassette. Mating-type is switched by Rad51-driven gene conversion of mat1 using a heterochromatic donor cassette, mat2-P or mat3-M . The Swi2-Swi5 complex, a mating-type switching factor, is central to this process by designating a preferred donor in a cell-type-specific manner. Swi2-Swi5 selectively enables one of two cis acting recombination enhancers, SRE2 adjacent to mat2-P or SRE3 adjacent to mat3-M . Here, we identified two functionally important motifs in Swi2, a Swi6 (HP1 homolog)-binding site and two DNA-binding AT-hooks. Genetic analysis demonstrated that the AT-hooks were required for Swi2 localization at SRE3 to select the mat3-M donor in P cells, while the Swi6-binding site was required for Swi2 localization at SRE2 to select mat2-P in M cells. In addition, the Swi2-Swi5 complex promoted Rad51-driven strand exchange in vitro . Taken together, our results show how the Swi2-Swi5 complex would localize to recombination enhancers through a cell-type specific binding mechanism and stimulate Rad51-driven gene conversion at the localization site.

Mating-type (P or M) of fission yeast Schizosaccharomyces pombe is determined by the transcriptionally active mat1 cassette and is switched by gene conversion using a donor, either mat2 or mat3, located in an adjacent heterochromatin region (mating-type switching; MTS). In the switching process, heterochromatic donors of genetic information are selected based on the P or M cell type and on the action of two recombination enhancers, SRE2 promoting the use of mat2-P and SRE3 promoting the use of mat3-M, leading to replacement of the content of the expressed mat1 cassette. Recently, we found that the histone H3K4 methyltransferase complex Set1C participates in donor selection, raising the question of how a complex best known for its effects in euchromatin controls recombination in heterochromatin. Here, we report that the histone H2BK119 ubiquitin ligase complex HULC functions with Set1C in MTS, as mutants in the shf1, brl1, brl2 and rad6 genes showed defects similar to Set1C mutants and belonged to the same epistasis group as set1Δ. Moreover, using H3K4R and H2BK119R histone mutants and a Set1-Y897A catalytic mutant, we found that ubiquitylation of histone H2BK119 by HULC and methylation of histone H3K4 by Set1C are functionally coupled in MTS. Cell-type biases in MTS in these mutants suggested that HULC and Set1C inhibit the use of the SRE3 recombination enhancer in M cells, thus favoring SRE2 and mat2-P. Consistent with this, imbalanced switching in the mutants was traced to compromised association of the directionality factor Swi6 with the recombination enhancers in M cells. Based on their known effects at other chromosomal locations, we speculate that HULC and Set1C control nucleosome mobility and strand invasion near the SRE elements. In addition, we uncovered distinct effects of HULC and Set1C on histone H3K9 methylation and gene silencing, consistent with additional functions in the heterochromatic domain.

Mating-type (P or M) of fission yeast Schizosaccharomyces pombe is determined by the transcriptionally active mat1 cassette and is switched by gene conversion using a donor, either mat2 or mat3 , located in an adjacent heterochromatin region (mating-type switching; MTS). In the process, heterochromatic donors of genetic information are selected based on the P or M cell type and on the action of two recombination enhancers, SRE2 promoting the use of mat2-P and SRE3 promoting the use of mat3-M , leading to replacement of the content of the expressed mat1 cassette. Recently, we found that the histone H3K4 methyltransferase complex Set1C participates in donor selection, raising the question of how a complex best known for its effects in euchromatin controls recombination in heterochromatin. Here, we report that the histone H2BK119 ubiquitin ligase complex HULC functions with Set1C in MTS, as mutants in the shf1, brl1, brl2 and rad6 genes showed defects similar to Set1C mutants and belonged to the same epistasis group as set1 Δ. Moreover, using H3K4R and H2BK119R histone mutants and a Set1-Y897A catalytic mutant indicated that ubiquitylation of histone H2BK119 by HULC and methylation of histone H3K4 by Set1C are functionally coupled in MTS. Cell-type biases in MTS in these mutants suggested that HULC and Set1C inhibit the use of the SRE3 recombination enhancer in M cells, thus favoring SRE2 and mat2-P . Consistently, imbalanced switching in the mutants was traced to compromised association of the directionality factor Swi6 with the recombination enhancers in M cells. Based on their known effects at other chromosomal locations, we speculate that HULC and Set1C might control nucleosome mobility and strand invasion near the SRE elements. In addition, we uncovered distinct effects of HULC and Set1C on histone H3K9 methylation and gene silencing, consistent with additional functions in the heterochromatic domain.

During homologous recombination, Rad51 forms a nucleoprotein filament on single-stranded DNA to promote DNA strand exchange. This filament binds to double-stranded DNA (dsDNA), searches for homology, and promotes transfer of the complementary strand, producing a new heteroduplex. Strand exchange proceeds via two distinct three-strand intermediates, C1 and C2. C1 contains the intact donor dsDNA whereas C2 contains newly formed heteroduplex DNA. Here, we show that the conserved DNA binding motifs, loop 1 (L1) and loop 2 (L2) in site I of Rad51, play distinct roles in this process. L1 is involved in formation of the C1 complex whereas L2 mediates the C1–C2 transition, producing the heteroduplex. Another DNA binding motif, site II, serves as the DNA entry position for initial Rad51 filament formation, as well as for donor dsDNA incorporation. Our study provides a comprehensive molecular model for the catalytic process of strand exchange mediated by eukaryotic RecA-family recombinases.

During homologous recombination, Rad51 forms a nucleoprotein filament on single-stranded DNA to promote DNA strand exchange. This filament binds to double-stranded DNA (dsDNA), searches for homology, and promotes transfer of the complementary strand, producing a new heteroduplex. Strand exchange proceeds via two distinct three-strand intermediates, C1 and C2. C1 contains the intact donor dsDNA whereas C2 contains newly formed heteroduplex DNA. Here, we show that conserved DNA binding motifs, loop 1 (L1) and loop 2 (L2) in site I of Rad51, play distinct roles in this process. L1 is involved in formation of the C1 complex whereas L2 mediates the C1-C2 transition, producing the heteroduplex. Another DNA binding motif, site II, serves as the DNA entry position for initial Rad51 filament formation, as well as for second donor dsDNA incorporation. Our study provides a comprehensive molecular model for the catalytic process of strand exchange mediated by eukaryotic RecA family recombinases.

In eukaryotes, all DNA transactions happen in the context of chromatin that often takes part in regulatory mechanisms. In particular, chromatin structure can regulate exchanges of DNA occurring through homologous recombination. Few systems have provided as detailed a view on this phenomenon as mating-type switching in yeast. Mating-type switching entails the choice of a template for the gene conversions of the expressed mating-type locus. In the fission yeast Schizosaccharomyces pombe, correct template choice requires two competing small recombination enhancers, SRE2 and SRE3, that function in the context of heterochromatin. These two enhancers act with the Swi2/Swi5 recombination accessory complex to initiate strand exchange in a cell-type-specific manner, from SRE2 in M cells and SRE3 in P cells. New research indicates that the Set1C complex, responsible for H3K4 methylation, and the Brl2 ubiquitin ligase, that catalyzes H2BK119 ubiquitylation, participate in the cell-type-specific selection of SRE2 or SRE3. Here, we review these findings, compare donor preference in S. pombe to the distantly related budding yeast Saccharomyces cerevisiae, and contrast the positive effects of heterochromatin on the donor selection process with other situations, where heterochromatin represses recombination.